1. Field of the Invention
The present invention generally relates to a system and method for desorption and ionization of analytes in a medium. More particularly, the present invention relates to a system and method for infrared matrix-assisted laser desorption and ionization mass spectrometry of analytes in a medium such as polyacrylamide gels.
2. Description of the Related Art
The recent sequencing of the human genome is accelerating the need to understand the variety, characteristics, and functions of the numerous proteins expressed by the genomes of humans and other organisms in response to internal and external stimuli. The characterization of proteins, such as their structure and function, is commonly referred to as proteomics. Mass spectrometry (xe2x80x9cMSxe2x80x9d) has proven to be an invaluable tool for proteomics, making possible the accurate profiling of proteins, polypeptides, peptides and other factors by precision measurements of molecular ion masses. Polyacrylamide gel electrophoresis (xe2x80x9cPAGExe2x80x9d) and related electrophoretic techniques are invaluable tools for the analysis of proteins, nucleic acids and other factors. Using PAGE techniques coupled with experimental manipulation and/or labeling techniques, it is possible to characterize structurally and functionally a variety of cellular determinants.
The ability to combine the preliminary separation provided by PAGE and resolution of MS into a coupled PAGE-MS system is highly desirable. Specifically, matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is generally acknowledged to be an integral part of any integrated strategy for proteomics, as discussed in the article xe2x80x9cProteomics: quantitative and physical mapping of cellular proteins,xe2x80x9d Blackstock et al., Trends in Biotech 17: 121-127 (1999). However, previous attempts to examine proteins by PAGE-MS or MALDI-MS required one or more of following limitations: (1) extraction of the protein from the polyacrylamide gel followed by routine sample preparation for MS; (2) transfer of the protein from the gel onto a membrane and permeation of the sample and membrane with a matrix for MALDI-MS; and/or (3) dehydration of the polyacrylamide gel and permeation or coating of the gel with a MALDI matrix.
The article xe2x80x9cMass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide electrophoresis gels,xe2x80x9d Cohen et al., Anal. Biochem. 247(2): 257-267 (1997), describes the use of mass spectrometry in the analysis of proteins eluted from SDS-PAGE gels using a 4-HCCA matrix and a fixed-wavelength laser operating in the ultraviolet range.
Researchers have described a method for identifying proteins from two-dimensional gels by electroblotting the proteins from the gels onto a membrane in the article xe2x80x9cIdentifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases,xe2x80x9d Henzel et al., Proc. Natl. Acad. Sci. USA 90: 5011-5015 (1993). The proteins were located by staining with Coomassie brilliant blue. Protein spots of interest were eluted from the membrane, trypsin digested, and analyzed by capillary high-performance liquid chromatography (HPLC), peptide mapping, and automated protein sequencing using the Edmond method. Mass spectrometry was performed by reconstituting an aliquot of the tryptic digest with a MALDI matrix and applying the sample to a mass spectrometer probe tip. The laser for the mass spectrometer was operated in the ultraviolet range at 337 nanometers (nm). The proteins and peptide fragments were identified by searching for the mass spectra of the ionization products in peptide fragment and protein sequence databases.
A technique described in xe2x80x9cMatrix-assisted laser desorption ionization mass-spectrometry of proteins electroblotted after polyacrylamide-gel electrophoresis,xe2x80x9d Strupat et al., Anal. Chem. 66: 464-470 (1994), was used to obtain MS signals of proteins physically transferred from a polyacrylamide gel to a membrane, wherein the samples were then treated with succinic acid, using a 100-nanosecond (ns) pulse from an Er:YAG laser operated at a fixed wavelength of 2.94 micrometer (xcexcm). These researchers attempted to do MALDI-MS, using an Er: YAG laser, directly from the polyacrylamide gel, both with and without the addition of exogenous matrix, but were unsuccessful.
Additional publications describe minor variations of these same techniques. The article xe2x80x9cCharacterization of SDS-PAGE-separated proteins by matrix-assisted laser desorption/ionization mass spectrometry,xe2x80x9d Liang et al., Anal. Chem. 68: 1012-1018 (1996), describes MALDI-MS performed on proteins on nitrocellulose membranes using a frequency-tripled Nd:YAG laser operating at a fixed wavelength of 355 nm.
The paper xe2x80x9cMass spectrometry of proteins directly from polyacrylamide gels,xe2x80x9d Ogorzalek Loo et al., Anal. Chem. 68:1910-1917 (1996), described preparing a protein sample on a polyacrylamide gel, identifying bands by staining or comparison to an identical stained gel, dehydrating the gel, adding a MALDI matrix (sinapinic acid) to gel positions of interest, irradiating the MALDI matrix with 337 nm laser light, and analyzing the products in a time-of-flight (TOF) mass spectrometer. The same group of researchers later described a variant of MALDI-MS performed on slices of polyacrylamide gel soaked in sinapinic acid in the article xe2x80x9cHigh sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins,xe2x80x9d Loo et al., Electrophoresis 20: 743-748 (1999).
Among other disadvantages, all of these techniques at least require considerable sample handling procedures that are impractical for adaptation to future designs for high-throughput sample analysis procedures. Therefore, there exists a need for an improved system and method for desorption and ionization of analytes in a medium. In particular, there is a need for an improved system and method that can allow direct high-speed sampling and analysis of proteins from polyacrylamide and other electrophoretic gels.
The present invention is related to a system and method for desorption and ionization of analytes in an ablation medium. In one aspect of the present invention, there is provided a method for desorption and ionization of analytes including the steps of preparing a sample comprising analytes in a medium having at least one component, selecting a resonant vibrational mode of at least one component of the medium, selecting a laser tuned to emit light substantially at the wavelength of the selected vibrational mode, and irradiating the sample with the laser light to cause medium ablation and desorption and ionization of the analytes. The method further includes the steps of passing the ionized analytes through a mass spectrometer, and obtaining a mass spectrum of the ionized analytes. The medium can be an electrophoresis medium that is in the form of an electrophoresis gel. The medium may also be chosen from, but is not limited to, materials such as cellulose acetate, paper, agarose an the like.
In another aspect, the present invention relates to a method for desorption and ionization of analytes including the steps of preparing a sample having analytes and a polyacrylamide medium having at least one component, selecting a resonant vibrational mode of at least one component of the medium, selecting a laser tuned to emit light substantially at the wavelength of the selected vibrational mode, and irradiating the sample with laser light to cause medium ablation and desorption and ionization of the analytes. In one embodiment, the sample is irradiated by laser light delivered in pulses, each pulse having duration of less than 5.0 picoseconds (ps), where the pulses are separated in time by more than 100 ps. The method further includes the steps of passing the ionized analytes through a mass spectrometer, and obtaining a mass spectrum of the ionized analytes.
In a further aspect, the present invention relates to a method for desorption and ionization of analytes including the steps of preparing a sample having analytes in a medium including at least one component, freezing the sample at a sufficiently low temperature so that at least part of the sample has an increase in viscosity and a decrease in vapor pressure, and irradiating the frozen sample with short-pulse radiation to cause medium ablation and desorption and ionization of the analytes. The method further includes the steps of selecting a resonant vibrational mode of at least one component of the medium and selecting an energy source tuned to emit short-pulse radiation substantially at the wavelength of the selected resonant vibrational mode.
In one embodiment, the energy source is a laser, where the laser can be a free electron laser tunable to generate short-pulse radiation. Alternatively, the laser can be a solid state laser tunable to generate short-pulse radiation. Moreover, the laser can be a gas laser or a metal vapor laser. The laser can emit light at various ranges of wavelength including a range of wavelength greater than 4.5 xcexcm and less than 10.0 xcexcm, a range of wavelength greater than 5.7 xcexcm and less than 6.5 xcexcm, a range of wavelength greater than 6.7 xcexcm and less than 7.3 xcexcm, and/or a range of wavelength greater than 7.3 xcexcm and less than 9.8 xcexcm.
In another embodiment, a resonant vibrational mode of at least one component of the medium can be selected from an absorption spectrum of the medium such as a Fourier-transform infrared absorption spectrum of the medium. The medium can be an electrophoresis medium that includes polyacrylamide. The medium can be in the form of a gel. The medium can also be chosen from materials such as cellulose acetate, paper, agarose and the like.
In yet another embodiment, by placing the sample in a sample support and immersing the sample support in liquid nitrogen for a period of time the sample may be cooled or frozen at a sufficiently low temperature such that any water within the sample may undergo a phase transition to change to ice.
In another embodiment, the sample may be prepared by spatially separating the analytes within a medium by electrophoresis. Moreover, the sample may be irradiated by sequentially irradiating a plurality of positions within the sample, wherein at least two irradiated positions correspond to the locations of the spatially separated analytes. Each of the plurality of positions is irradiated by radiation delivered in pulses, each pulse having a duration of less than the relaxation time of a selected vibrational mode of at least one component of the medium. The pulses are separated in time by intervals, each interval having a duration of at least ten times the relaxation time of the selected vibrational mode. Alternatively, each pulse may have a duration of less than the thermal or mechanical relaxation time of the at least one component of the medium. In one embodiment, moreover, the sample is moved in a motion back and forth relative to the laser light to form a rastering trace such that the desorption and ionization of the analytes occur substantially at a same region in space such as a region around the focal point of the laser light.
In yet another aspect, the present invention relates to a system for desorption and ionization of analytes. The system includes means for preparing a sample comprising analytes in a medium having at least one component, means for selecting a resonant vibrational mode of at least one component of the medium, means for emitting light substantially at the wavelength of the selected vibrational mode, and means for irradiating the sample to cause medium ablation and desorption and ionization of the analytes. The system further includes means for freezing the sample to a sufficiently low temperature so that at least part of the sample has an increase in viscosity and a decrease in vapor pressure, and means for delivering light in pulses, each pulse having a duration of less than the relaxation time of the selected vibrational mode, wherein the pulses are separated in time by intervals, each interval having a duration of at least ten times the relaxation time of the selected vibrational mode. Alternatively, each pulse may have a duration of less than the thermal or mechanical relaxation time of the at least one component of the medium. In one embodiment, moreover, the sample is moved back and forth relative to the laser light to form a rastering trace such that the desorption and ionization of the analytes occur substantially at a same region in space such as a region around the focal point of the laser light.
In a further aspect, the present invention relates to a system for desorption and ionization of analytes. The system includes means for preparing a sample having analytes and a polyacrylamide medium having at least one component, means for selecting a resonant vibrational mode of at least one component of the medium, means for emitting light substantially at the wavelength of the selected vibrational mode, and means for irradiating the sample with laser light to cause medium ablation and desorption and ionization of the analytes. Moreover, the system includes means for stabilizing the sample for compatibility with high-vacuum conditions and means for delivering light in pulses, each pulse having a duration of less than the relaxation time of the selected vibrational mode, wherein the pulses are separated in time by intervals, each interval having a duration of at least ten times the relaxation time of the selected vibrational mode. Alternatively, each pulse may have a duration of less than the thermal or mechanical relaxation time of the at least one component of the medium. In one embodiment, moreover, the sample is moved in a motion back and forth relative to the laser light to form a rastering trace such that the desorption and ionization of the analytes occur substantially at a same region in space such as a region around the focal point of the laser light.
In yet a further aspect, the present invention relates to a system for desorption and ionization of analytes. The system includes means for preparing a sample having analytes in a medium including at least one component, means for freezing the sample to a sufficiently low temperature so that at least part of the sample has an increase in viscosity and a decrease in vapor pressure, and means for irradiating the chilled sample with short-pulse radiation to cause medium ablation and desorption and ionization of the analytes. Additionally, the system includes means for selecting a resonant vibrational mode of at least one component of the medium, and means for tuning an energy source to emit short-pulse radiation substantially at the wavelength of the selected resonant vibrational mode.
In yet another aspect, the present invention relates to a system for desorption and ionization of analytes. The system includes a support for holding a sample of analytes in a medium, a laser source emitting light corresponding to a selected vibrational mode of at least one component of the medium, and a plurality of optics elements directing the emitted light to irradiate the sample to cause medium ablation and desorption and ionization of the analytes, an ion accelerator for injecting the ionized analytes into a mass spectrometer, a mass spectrometer which separates the accelerated ionized analytes according to their masses, a detector for the mass determination of ionized analytes separated according to their masses, data collection equipment for recording of the spectrum of determined masses, and data presentation equipment for displaying of the spectrum of determined masses. Additionally, the system includes means for selecting a resonant vibrational mode of at least one component of the medium, and means for tuning an energy source to emit short-pulse radiation substantially at the wavelength of the selected resonant vibrational mode.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.